ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10077-3244
Journal of South Asian Association of Pediatric Dentistry
Volume 5 | Issue 3 | Year 2022

Comparative Evaluation of Triple Organic Paste vs Triple Antibiotic Paste: An In Vitro Study

Sudheer Kumar Kotha1, BV Thimma Reddy2, Uday Chowdary Birapu3, Hemanth Kumar L Raichurkar4, Kumar J Raghavendra5

1,4,5Department of Pedodontics & Preventive Dentistry, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, India

2Department of Pedodontics, Panneya Dental College, Hyderabad, Telangana, India

3Department of Pedodontics & Preventive Dentistry, Lenora Institute of Dental Sciences, Rajamahendravaram, Andhra Pradesh, India

Corresponding Author: Sudheer Kumar Kotha, Department of Pedodontics & Preventive Dentistry, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, India, Phone: +91 8919474593, e-mail: kothasudheerkumar266@gmail.com

Received on: 16 September 2022; Accepted on: 27 October 2022; Published on: 26 December 2022

ABSTRACT

Context: The concept of the revascularization technique mainly depends on root canal sterilization, the presence of a template (blood clot), and coronary filling, which prevents marginal leakage. Successful disinfection is carried out by using triple antibiotic paste (TAP) as an intracanal medication due to its excellent antimicrobial activity, but its use is associated with discoloration of the tooth crown, the development of bacterial resistance, and allergic reactions.

Aims: To evaluate the antibacterial efficacy of triple organic paste vs TAP against strains of Enterococcus faecalis, Streptococcus mutans, and Candida albicans.

Methods and material: Natural products were divided into six groups and further divided into 18 subgroups. Minimum inhibitory concentration (MIC) was determined for each group using the trial and error method, and results were obtained after incubation for 24 hours at 37°C. Antibacterial efficacy was determined by zone of inhibition and compared with TAP (control group) individually against E. faecalis, S. mutans, and C. albicans. The values were recorded and subjected to statistical analysis by one-way analysis of variance (ANOVA) and Tukey’s post hoc test.

Results: All the subgroups have shown significant zone of inhibition values against E. faecalis, S. mutans, and C. albicans, except for the subgroup IC, where no statistically significant values were found when compared with the control group.

Conclusion: Most of the combinations of natural products have shown better antibacterial efficacy when compared with TAP, suggesting its use to overcome the disadvantages of TAP. Further clinical studies are required for evaluating the antibacterial efficacy in vivo.

How to cite this article: Kotha SK, Reddy BVT, Birapu UC, et al. Comparative Evaluation of Triple Organic Paste vs Triple Antibiotic Paste: An In Vitro Study. J South Asian Assoc Pediatr Dent 2022;5(3):136-146.

Source of support: Nil

Conflict of interest: None

Keywords: Apexification, Crown discoloration, Regenerative endodontic treatment, Triple antibiotic paste, Triple organic paste

INTRODUCTION

Eradication of the root canal infection is very important in the revascularization procedure, which includes the use of various techniques of instrumentation, irrigation protocols, and intracanal medicated dressings.1 Considering the multifaceted nature of tooth infection, TAP consisting of metronidazole, ciprofloxacin, and minocycline has been proposed as a root canal medicament owing to its antimicrobial effects.2 But the use of it is associated with the development of crown discoloration and multidrug bacterial resistance, which has paved the way for the usage of biologically safe herbal alternatives.3

RATIONALE

Attempt to compare the antimicrobial efficacy of TAP and various formulations of herbal medicaments on strains of E. faecalis, C. albicans, and S. mutans.

AIM

To compare the antimicrobial efficacy of triple organic paste vs TAP.

OBJECTIVES

To evaluate the antimicrobial efficacy of different combinations of six herbal medicaments against microbial strains of the following:

Subjects and Methods

For this study, crude extracts of the following herbal products were procured (Figs 1 and 2), namely from neem leaves, turmeric rhizomes, ginger rhizomes, garlic bulbs, bitter gourd seeds, and triphala.

Figs 1A to C: Components of triple antibiotic paste: (A) Cifran 250 mg; (B) Metrogyl 200 mg; (C) Minoz 100 mg

Fig. 2: Crude extracts of natural products

Crude extracts of herbal medicaments procured from the Indian raw herbs website and prepared in the Department of Microbiology, SVS Institute of Dental Sciences.

(https://indianjadibooti.com/Jadistore/?gclid=Cj0KCQiAhf2MBhDNARIsAKXU5GRAcEl7d8-I7S8jJCVAEi3oXUOum_u16SVOEkpAF8QeWP8oFsmYRjQaAgfyEALw_wcB.)

Bacterial cultures of E. faecalis, C. albicans, and S. mutans [Microbial Type Culture Collection and Gene Bank strain (MTCC)—497] (Fig. 3) were procured from MTCC India.

Figs 3A and B: Bacterial cultures: (A) E. faecalis; (B) S. mutans

METHODOLOGY

The sample was divided into a control group and an experimental group. The experimental group was categorized into six groups (Flowchart 1).

Flowchart 1: Categorization of sample

These were further divided into subgroups based on combinations of three herbs without repetition of any three combinations; they are tabulated in Table 1.

Table 1: Showing combinations of herbal products
Groups Subgroups
Neem (N) IA) N, T, Gi IB) N, T, Ga IC) N, T, Bi
Turmeric (T) IIA) T, Gi, Ga IIB) T, Ga, Bi IIC) T, Bi, Tr
Ginger (Gi) IIIA) Gi, Ga, Bi IIIB) Gi, Bi, Tr IIIC) Gi, Ga, Tr
Garlic (Ga) IVA) Ga, Bi, Tr IVB) Ga, N, Gi IVC) Ga, Bi, N
Bitter gourd (Bi) VA) Bi, Tr, N VB) Bi, N, Gi VC) Bi, T, Gi
Triphala (Tr) VIA) Tr, N, Ga VIB) Tr, N, Gi VIC) Tr, T, Ga

Preparation of Tap (Control Group)

Triple antibiotic powder preparation was done in the Department of Pharmacology (SVS Institute of Dental Sciences, Mahbubnagar, Telangana, India), where commercially available drugs in tablet form were taken—ciprofloxacin (cifran 250 mg), metronidazole (metrogyl 400 mg), and minocycline (minoz 100 mg) (Ranbaxy Laboratories Pvt Ltd, India) (Fig. 1). Just before mixing they were dispensed on a mixing slab in the ratio of 1:1:1 and titrated with distilled water as a vehicle to achieve consistency of a paste (Figs 4 and 5).

Figs 4A and B: MIC for E. faecalis Group I: (A) Neem (preincubation); (B) Neem (postincubation)

Fig. 5: Preparation of triple antibiotic powder

Determination of MIC for Natural Products (Experimental Group)

Prior to performing MIC, all these natural powders were sterilized at 37°C for 45 minutes in a hot air oven and were diluted using nutrient agar broth and subjected to incubation at 37°C for 24 hours.

Minimum inhibitory concentration (MIC) is done individually for each of the bacterial strains using these six natural products by trial and error method (Fig. 4). For individual natural product groups for each of the bacterial strains, values are recorded and noted. Based on these values, MIC values for different subgroup combinations were calculated (Table 2).

Table 2: MIC values
E. faecalis C. albicans S. mutans
Neem 146 mg/µL 91 mg/µL 37 mg/µL
Turmeric 91 mg/µL 110 mg/µL 37 mg/µL
Ginger 91 mg/µL 55 mg/µL 55 mg/µL
Garlic 91 mg/µL 92 mg/µL 37 mg/µL
Bitter gourd 104 mg/µL 73 mg/µL 73 mg/µL
Triphala 104 mg/µL 110 mg/µL 73 mg/µL

Preparation of Culture Plates

Three bacterial strains, each about 50 µL were taken using a micropipette onto Petri dishes loaded with blood agar media for S. mutans, E. faecalis, and Sabouraud dextrose agar for C. albicans by lawn streaking method over the entire surface of the plates. Then four wells of 7 × 4 mm (size × depth) were made under aseptic conditions to dispense the medicaments. TAP was mixed using distilled water as a vehicle, and combinations of natural products were mixed using distilled water based on MIC values and dispensed into wells of culture plates (Fig. 6).

Figs 6A and B: (A) Preparation of wells; (B) Dispensing medicament into a culture plate

Parameter Evaluated

Diameter of zone of inhibition.

Microbiological Analysis

Culture plates with medicaments were subjected to incubation at 37°C for 24 hours in aerobic conditions and evaluated for the zone of inhibition (Figs 7 to 11). The diameter of zones of inhibition was measured by using digital vernier calipers in four different directions and the mean value of it was taken as the diameter of the zone of inhibition (Figs 8 and 10).

Figs 7A and B: Zone of inhibition for E. faecalis: (A) (Preincubation) group I, group II, group III, group IV, group V, and group VI; (B) (Postincubation) group I, group II, group III, group IV, group V, and group VI

Fig. 8: Measurement of zone of inhibition using digital vernier calipers

Figs 9A and B: Zone of inhibition for C. albicans: (A) (Preincubation) group I, group II, group III, group IV, group V, and group VI; (B) (Postincubation) group I, group II, group III, group IV, group V, and group VI

Fig. 10: Measurement of zone of inhibition using digital vernier calipers

Figs 11A and B: Zone of inhibition for S. mutans: (A) (Preincubation) group I, group II, group III, group IV, group V, and group VI; (B) (Postincubation) group I, group II, group III, group IV, group V, and group VI

RESULTS

The results were tabulated and data were subjected to statistical analysis.

Statistical test applied: one-way ANOVA and Tukey’s post hoc test.

Results have shown that the mean diameter of the zone of inhibition for the control group and all the subgroups, except for the subgroup IC, have shown statistically significant values when tested for E. faecalis, C. albicans, and S. mutans, respectively (Tables 3 to 5).

Table 3: Intergroup comparison of zones of inhibition (mm) for all the groups when tested for E. faecalis
Mean Standard deviation (SD) p-value Significant pairs
Group I (E. faecalis) Control (1) 35.0000 0.81650 0.000 HS 1–2; 1–3; 1–4; 2–3; 2–4
Group IA (2) 25.2500 1.50000
Group IB (3) 16.7500 1.25831
Group IC (4) 17.2500 0.95743
Group II (E. faecalis) Control (1) 38.7500 1.50000 0.000 HS 1–3; 1–4; 2–3; 2–4; 3–4
Group IIA (2) 36.7500 0.95743
Group IIB (3) 27.0000 1.15470
Group IIC (4) 17.0000 0.00000
Group III (E. faecalis) Control (1) 29.0000 0.81650 0.000 HS 1–2; 1–3; 1–4; 2–3; 2–4; 3–4
Group IIIA (2) 30.7500 0.95743
Group IIIB (3) 18.7500 0.95743
Group IIIC (4) 23.0000 0.00000
Group IV (E. faecalis) Control (1) 27.2500 0.95743 0.000 HS 1–2; 1–3; 1–4; 2–3; 3–4
Group IV (2) 20.0000 0.00000
Group IVB (3) 17.0000 0.81650
Group IVC (4) 19.2500 0.95743
Group V (E. faecalis) Control (1) 29.2500 0.95743 0.000 HS 1–2; 1–3; 1–4; 2–3; 3–4
Group VA (2) 18.5000 1.91485
Group VB (3) 13.0000 0.81650
Group VC (4) 19.2500 0.95743
Group VI (E. faecalis) Control (1) 25.2500 1.70783 0.000 HS 1–2; 1–3; 1–4
Group VIA (2) 19.0000 1.15470
Group VIB (3) 17.7500 0.50000
Group VIC (4) 17.2500 0.95743

Statistical test applied, one-way ANOVA and Tukey’s test (post hoc); HS, high statistical significance at p < 0.01

Table 4: Intergroup comparison of zones of inhibition (mm) for all the groups when tested for C. albicans
Mean SD p-value Significant pairs
Group I (C. albicans) Control (1) 22.5000 1.29099 0.000 HS 1–2; 1–3; 1–4
Group IA (2) 15.2500 1.25831
Group IB (3) 16.2500 1.25831
Group IC (4) 16.7500 2.06155
Group II (C. albicans) Control (1) 26.2500 2.06155 0.000 HS 1–2; 1–3; 1–4; 2–4; 3–4
Group IIA (2) 15.7500 0.95743
Group IIB (3) 15.0000 0.00000
Group IIC (4) 31.0000 1.41421
Group III (C. albicans) Control (1) 24.5000 0.57735 0.000 HS 1–2; 1–3; 1–4; 2–3; 2–4
Group IIIA (2) 19.0000 0.81650
Group IIIB (3) 32.2500 0.95743
Group IIIC (4) 33.7500 0.95743
Group IV (C. albicans) Control (1) 24.5000 3.10913 0.000 HS 1–2; 1–3; 2–3; 2–4
Group IVA (2) 28.7500 0.95743
Group IVB (3) 17.5000 1.29099
Group IVC (4) 21.2500 1.25831
Group V (C. albicans) Control 22.2500 1.70783 0.000 HS
Group VA 34.0000 0.81650
Group VI (C. albicans) Control (1) 25.5000 1.00000 0.000 HS 1–2; 1–3; 1–4; 2–4
Group VIA (2) 31.2500 0.95743
Group VIB (3) 30.0000 0.81650
Group VIC (4) 28.2500 1.25831

Statistical test applied, one-way ANOVA; HS, high statistical significance at p < 0.01

Table 5: Intergroup comparison of zones of inhibition (mm) for all the groups when tested for S. mutans
Mean SD p-value Significant pairs
Group I (S. mutans) Control(1) 22.7500 1.25831 0.000 HS 1–4; 2–3, 2–4, 3–4
Group IA (2) 25.2500 1.25831
Group IB (3) 21.2500 1.50000
Group IC (4) 17.0000 0.00000
Group II (S. mutans) Control (1) 48.2500 2.50000 0.000 HS 1–3; 1–4; 2–3; 2–4; 3–4
Group IIA (2) 47.2500 0.95743
Group IIB (3) 41.5000 1.29099
Group IIC (4) 33.7500 1.50000
Group III (S. mutans) Control (1) 54.2500 0.50000 0.000 HS 1–2; 1–3; 1–4; 2–4
Group IIIA (2) 33.2500 1.50000
Group IIIB (3) 34.2500 1.50000
Group IIIC (4) 36.0000 0.81650
Group IV (S. mutans) Control (1) 54.2500 1.25831 0.000 HS 1–2; 1–3; 1–4; 2–3; 2–4; 3–4
Group IVA (2) 22.5000 1.00000
Group IVB (3) 17.0000 0.81650
Group IVC (4) 14.2500 0.50000
Group V (S. mutans) Control (1) 61.2500 2.21736 0.000 HS 1–2; 1–3; 1–4; 2–3; 2–4; 3–4
Group VA (2) 26.2500 0.50000
Group VB (3) 18.5000 1.00000
Group VC (4) 33.2500 0.50000
Group VI (S. mutans) Control (1) 56.2500 1.70783 0.000 HS 1–2; 1–3; 1–4
Group VIA (2) 19.7500 0.50000
Group VIB (3) 21.7500 0.50000
Group VIC (4) 21.0000 0.81650

Statistical test applied, one-way ANOVA and Tukey’s test (post hoc); HS, high statistical significance at p < 0.01

DISCUSSION

Root canal obturation for immature permanent teeth with large open apex is challenging even for the most experienced clinician without extending the filling beyond the apex into the periapical tissues.

The history of revascularization technique dates back to the early 1960s, when studies conducted by Birger Nygaard-Östby showed that new vascularized tissue can be induced in the apical third of the root end of young, immature teeth with necrotic pulp and apical lesion by inducing a blood clot in the apical third of a sterilized root canal by instrumenting beyond apex, using an endodontic file before root canal filling.4 Later, in 2001, Iwaya et al. introduced the term revascularization, based on a study performed on a necrotic immature mandibular second premolar with chronic apical abscess.5

These findings paved the way for the clinical protocol of revascularization in infected immature teeth, which helps in pulp regeneration and promotes root-end closure.4,6

The success of pulp revascularization depends on three elements: root canal sterilization, the presence of a template (blood clot), and coronary filling, which prevents marginal leakage.7

In revascularization procedures, TAP has been recommended as an intracanal medication owing to its excellent polymicrobial activity in the infected root canals of immature permanent teeth.8,11 Saoud et al.5 later developed TAP, which consists of ciprofloxacin, minocycline, and metronidazole.12 Minocycline, which is a semisynthetic tetracycline derivative, is associated with the risk of crown discoloration13 because of chelation between minocycline and calcium ions to form insoluble complexes.14 This can be prevented by sealing the dentinal tubules of the pulp chamber (etching and bonding).15 The usage of TAP leads to the development of crown discoloration, bacterial resistance, and hypersensitivity reactions.16

In the present study, in order to overcome the above-mentioned systemic and local side effects of components of TAP, various natural products17 and their combinations have been tested as an alternative to TAP.

Natural products being one of the major sources of new drug molecules are derived from prokaryotic bacteria, eukaryotic microorganisms, plants, and various animal organisms. Recently, many studies have focused on the newly synthesized molecules as potential antimicrobial agents, which are derived from plant and microbial extracts, essential oils, and pure secondary metabolites.

Azadirachta indica—Neem

It is known as “Indian neem/margosa tree/Indian lilac.” Antimicrobial efficacy is mainly due to tetranortriterpenoid, which includes nimbin, nimbidinin, nimbolide, and nimbidinic acid, which has significant activity against many gram-positive and gram-negative microorganisms, such as S. mutans, Mycobacterium tuberculosis, streptomycin resistant strains, s. pyogenes (streptococcus pyogenes), Vibrio cholera, and Klebsiella pneumonia.

Bohora et al.18 conducted a study using aqueous and ethanolic extract of neem leaf and concluded that neem has potential action against E. faecalis, suggesting its use as a potential alternative to sodium hypochlorite (NaOCl) hypochlorite against endodontic pathogens.

Curcuma longa—Turmeric

It has been traditionally used as a medicine for treating sprains and swellings caused by injury. The main antimicrobial compounds include polyphenols like curcuminoids (curcumin, dimethoxycurcumin, and bisdemethoxycurcumin), among which curcumin is important. It destroys the assembly of a protein-filamenting temperature-sensitive mutant Z (FtsZ) protofilaments and enhances the guanosine triphosphate ase activity of FtsZ, leading to the destruction of the microbial cell wall.

Triphala

It is an ayurvedic combination of dried and powdered fruits of three different herbal plants, namely Terminalia bellirica (bibhitaki), T. chebula (halituki), and Emblica officinalis (Amalaki). Antibacterial efficacy is mainly due to tannins, quinones, flavonoids, gallic acid, and vitamin C, among which tannic acid is the most important.

A study conducted by Prabhakar et al.19 has shown that triphala was effective against root canal biofilms and is similar to NaOCl and doxycycline-based irrigating agents because of its free radical scavenging action preventing the formation of biofilm.

Allium sativum—Garlic

Since ancient times, garlic has found its use medicinally as an antimicrobial and antihyperlipidemic agent. The allicin, which is the active ingredient, has antimicrobial action on the cell wall and cell membrane of the microbes. Studies have shown that the ethanolic extract of garlic is effective against Staphylococcus aureus. A study conducted by Alrazhi et al.20 concluded that garlic extract reported inhibition of the formation of biofilms against S. epidermidis and E. faecalis.21

Zingiber officinale—Ginger

Active ingredients include zingerone, gingerol, and 6-shogaol, which have demonstrated significant antimicrobial activity against gram-negative bacteria like Porphyromonas gingivalis, P. endodontalis, and Prevotella intermedia.

Momordica charantia—Bitter gourd

A potential herbal plant used as a vegetable and medicine consisting of various medicinally important biochemicals like triterpene, protein, steroid, alkaloid, and phenolic compounds responsible for its biological and pharmacological activities.

A study conducted by Umesh et al.22 concluded that bitter gourd extract, when used in conjunction with chloroform, ethanol, and petroleum ether showed antibacterial activity against S. mutans and Lactobacillus.

All these herbal products are known to possess various medicinal properties, which include anti-inflammatory, antioxidant, anticarcinogenic, antimutagenic, antifungal, anticoagulant, antidiabetic, antihypertensive, antimicrobial, antifertility, antiviral, antihelminthic, antimalarial, anti-ulcerative, and immune protective effects.

Disk diffusion and agar dilution methods are routinely employed in laboratory methods to evaluate the in vitro antimicrobial activity of natural extracts.

Qualitative measurement of antibiotic resistance is done in a quick and easy way using a zone of inhibition test to measure and compare levels of inhibitory activity.

In the present study, the mean diameter of the zone of inhibition for the control group, IA and IB subgroups of group I, was statistically significant (p-value <0.01) when tested for E. faecalis, C. albicans, and S. mutans except for the subgroup IC. Mustafa.23 conducted a study assessing the antimicrobial efficacy of neem extract against E. faecalis, and concluded that neem leaf extract shows comparable zones of inhibition with that of chlorhexidine and NaOCl, suggesting its use as an alternative intracanal medicament.

For the second group, mean diameter of the zone of inhibition for the control group, IIA, IIB, and IIC subgroups was statistically significant when tested for E. faecalis, C. albicans, and S. mutans. Upadhyay et al.24 conducted a study to evaluate the antibacterial efficacy of C. longa against endodontic pathogens and concluded that a combination of turmeric oleoresin and calcium hydroxide had shown maximum inhibition of E. faecalis, Escherichia coli, and S. aureus, suggesting its use as intracanal medicament.

For the third group, the mean diameter of the zone of inhibition for the control group, IIIA, IIIB, and IIIC subgroups was statistically significant when tested for E. faecalis, C. albicans, and S. mutans. A study conducted by Abood and Witwit.25 concluded that the inhibitory effect of ginger oil was most effective on isolated bacteria when compared with amoxicillin and green tea.

For the fourth group, the mean diameter of the zone of inhibition for the control group, IVA, IVB, and IVC subgroups was statistically significant when tested for E. faecalis, C. albicans, and S. mutans. Beshr and Abdelrahim.26 conducted a study to evaluate the antibacterial activity of mineral trioxide aggregate (MTA) Fillapex and Guttaflow 2 sealers with chitosan and A. sativum against E. faecalis strain and concluded that incorporation of A. sativum and chitosan into MTA Fillapex sealer enhanced its antimicrobial efficacy against E. faecalis.

For the fifth group, the mean diameter of zone of inhibition for the control group, VA, VB, and VC subgroups was statistically significant when tested for E. faecalis, C. albicans, and S. mutans. Arora et al.27 conducted a study using herbal extracts as endodontic irrigants and concluded that bitter gourd showed the maximum zones of inhibition against E. faecalis and C. albicans.

For sixth group, the mean diameter of zone of inhibition for the control group, VIA, VIB, and VIC subgroups was statistically significant when tested for E. faecalis, C. albicans, and S. mutans. A study conducted by Bhargava et al. has shown that NaOCl has the highest antimicrobial activity when compared with neem, triphala, and green tea, respectively, as endodontic irrigant.28

On intergroup comparison, when tested for E. faecalis for all six groups, mean diameters of zone of inhibition were statistically significant in comparison with the control group (Table 3 and Fig. 12). On intergroup comparison, when tested for C. albicans for all the six groups, mean diameters of zone of inhibition were statistically significant in comparison with the control group (Table 4 and Fig. 13). On intergroup comparison, when tested for S. mutans for all the six groups, mean diameters of zone of inhibition were statistically significant in comparison with the control group (Table 5 and Fig. 14). Statistical tests used in this study are one-way ANOVA and Tukey’s post hoc test. The present study employed one-way ANOVA for intragroup comparison and Tukey’s post hoc test for intergroup comparison.

Fig. 12: Mean zone of inhibition for E. faecalis for the six groups

Fig. 13: Mean zone of inhibition for C. albicans for the six groups

Fig. 14: Mean zone of inhibition for S. mutans for the six groups

Limitations of the Study

Further laboratory studies, toxicology studies, and clinical studies are needed to be done to evaluate the antibacterial efficacy of the various combinations of herbal medicaments in comparison with TAP and before utilizing the extracts on a patient.

CONCLUSION

Within the limits of the study, the following conclusions could be drawn.

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